1. Field of the Invention
The present invention relates to multizone dry chemistry analytical elements for the determination of an analyte, such as albumin, in aqueous fluids, and to methods for the use of such elements. In particular, the invention relates to the continuous release within such elements of a chromogenic indicator reagent composition to a reaction zone to thereby eliminate or reduce interference from competing substances, if present.
2. Discussion Relative to the Prior Art
In the analytical determination for analytes in aqueous fluids such as urine and serum or other body fluids, chromogenic indicator reagent compositions that interact with such analytes to produce a color response are frequently employed. The color response is then compared with appropriate standards or calibrators to determine the quantity of analyte present in the fluid.
In many instances, interfering substances accompany the analytes and interact with the reagent composition to produce a color response bearing the same color absorption characteristics as those produced by the analyte reagent interaction. The analyte-reagent and interferent-reagent interactions are additive resulting in a biased overall color response from which the component attributable to analyte-reagent interaction cannot readily be discerned. For example, in the determination of magnesium in aqueous fluids by interaction with chromogenic indicators such as Eriochrome.RTM. Black T (EBT) or Titan yellow dye to produce a characteristic color response, calcium is a potential interferent. If present, calcium also interacts with either dye to produce a biased color response. Similarly, in the determination of albumin in body fluids by interaction of albumin with buffered chromogenic indicator reagents to produce a color response, accompanying protein interferents such as globulins or transferrin also interact with the reagent to produce a biased color response.
The concern for reducing interference to analyte assays using chromogenic indicator reagents applies to many assays. For the purpose of illustration, the prior art and background relative to albumin assays is set forth below.
Albumin is the most abundant of plasma proteins, generally constituting slightly over half of the total protein in the plasma. Albumin has a molecular weight of about 69,000, is synthesized in the liver, and has a half-life of about four weeks. It has two important roles:
(a) regulating the water balance between blood and tissues, and
(b) functioning as a transport molecule for various materials which are only slightly soluble in water, such as bilirubin, fatty acids, cortisol, thyroxine, and a number of drugs including sulfonamides and barbiturates.
It is frequently important to determine whether patients have a deficiency of serum albumin. A deficiency of albumin in a patient's serum indicates a possibility of edema, nephrosis, cirrhosis, multiple sclerosis, hepatitis and other disease states. Also, albumin deficiency interferes with the transport of insoluble materials. Thus, albumin levels in body fluids are a useful tool for diagnosing illness. Accordingly, there is a need for procedures and materials which detect and quantify albumin, particularly low values of albumin in body fluids.
The determination of albumin in fluids is now widely practiced using buffered bromcresol green (hereinafter referred to as BCG) solutions or test strips as disclosed in U.S. Pat. No. 3,533,749 issued Oct. 13, 1970 to N. Kleinman and U.S. Pat. No. 3,485,587 issued Dec. 23, 1969 to A. S. Keston. Bromcresol green (BCG) is a sulphonphthalein species of chromogenic indicator materials that exhibit a color response, i.e., a change in color, by dye-binding interaction with proteins such as albumin. Such color response is proportionally related to the amount of albumin present. As noted above, the indicators are buffered which insures against color response to pH changes to which the indicators are otherwise sensitive.
Chromogenic indicators such as BCG are not entirely specific to albumin. Globulins, transferrin and other proteins normally present, for example, in human biological fluids compete for dye binding with the indicator causing interference to albumin assays. The interference is particularly significant with albumin at low concentration levels. For example, the normal total protein content, including albumin and globulin, of human serum ranges from about 6-8 g/dL. Of this, about 2.9 g/dL represents globulins, the balance being predominantly albumin. In normal serum (i.e., from healthy individuals), the albumin-to-globulin ratio is about 1.6:1 while in disease states the ratio may drop to about 0.7:1. A useful clinical assay must therefore avoid binding with globulins and result in a selective binding with albumin.
Several authors have suggested solutions to the problem of non-specificity in BCG-based albumin determinations. In 1976, J. C. Gustafsson reported that the reaction of serum albumin with BCG is faster than the reaction of BCG with other proteins. (Gustafsson, J. E. C., Clin Chem., 22:616, 1976). Accordingly, Gustafsson proposed to measure the absorbance of the solution twice after serum is mixed with BCG reagent: immediately and at 60 minutes. The immediate reading when extrapolated back to zero minutes is then employed to determine the albumin concentration. (The sixty minute reading is employed in the determination of serum protein components in addition to albumin.) The success of this method is predicated on the capability of one being able to obtain an "immediate" absorbance reading, an impractical requirement for automated systems that have inherent time lags of one or more minutes before meaningful readings can be taken. The Gustafsson zero minute procedure, moreover, is applicable to solution assays wherein for example, 10 .mu.L of serum is interacted with 2.0 ml (2000 .mu.L) of BCG reagent solution, a 200-fold dilution. However, with the advent of dry chemistry analytical assay elements, dilution of samples and the use of space-consuming liquid reagents are no longer required. In use, such elements are contacted with undiluted body fluids to produce a color response. The present inventors have determined, in this regard, that in undiluted samples, the total protein-BCG color response, i.e., the response from both albumin and globulins, is significantly more intense than in solution assays. Accordingly, zero minute determination of albumin in dry elements using the procedure of Gustafsson is not only impractical, but less specific to albumin compared to solution assays.
D. Webster (Clin. Chem., 23:663, 1977) confirmed the non-specificity of the BCG-based albumin determination for solution assays. That author concluded that a reading taken within 30 seconds was specific to albumin when 3.0 g/liter is subtracted from the result obtained. As in the Gustafsson procedure, Webster's method is predicated on early read times and an assay in dilute solution (200-fold dilution of sample fluids). The use of an arbitrary constant as a correction factor assumes, moreover, that the albumin result is overstated from the outset and so signifies a non-specific assay. More important, it does not account for real variations from the arbitrary 3.0 g/liter factor.
After Webster and Gustafsson, the problem of non-specificity of the BCG-based albumin assay was further disclosed by Ingwersen, S. and Raabo, E., Clin. Chim, Acta, 88:545, 1978. Ingwersen at al. determined that in dilute systems such as those studied by Gustafsson and Webster, lowering the concentration of BCG in the reagent solution suppressed the binding of globulins to such a degree that a reading could be taken as late as one minute after addition of the reagent. This reading was reported to be almost entirely due to the albumin-BCG complex.
The method according to Ingwerson et al. permits a reading to be taken up to one minute after initiation of the assay in solution. However, the present inventors have determined that when BCG indicator is used in conventional dry analytical elements in amounts suggested by Ingwersen, severe interference from competing proteins such as globulins is encountered. Still further, the interference is observed immediately, i.e., within less than one minute, thus emphasizing the distinction between dilute solution systems and dry chemistry systems employing undiluted serum.
If one were to consider lowering further the amount of BCG indicator in dry analytical elements in order to reduce the aforementioned globulin interference, it would generally not be possible without loss in sensitivity and linearity of response to anticipated levels of albumin, particularly high (e.g., 5 g/dL or higher) albumin levels.
The use of analytical elements for the determination of protein is well known. In the aforementioned Keston U.S. Pat. No. 3,485,587, single zone elements that include absorbent paper impregnated with buffered indicator reagent such as BCG are proposed. In use, the element is saturated with a sample of body fluid causing instantaneous contact of all the reagent with fluid to initiate color development. Such elements are useful to detect the presence or estimate the quantity of total protein. They cannot, however, accurately selectively determine albumin in the presence of globulins because, as previously noted, globulins compete for available indicator.
Multizone elements for the determination of analytes (including albumin) are disclosed in U.S. Pat. No. 3,992,158 issued Nov. 16, 1976 to E. P. Przybylowicz and A. G. Millikan and U.S. Pat. No. 4,042,335 issued Aug. 16, 1977 to P. L. Clement. In U.S. Pat. No. 3,992,158, FIG. 2 therein depicts a porous spreading layer on a reagent layer overlying a support. Numerous matrix materials such as gelatin, agarose and water soluble vinyl polymers, are disclosed as useful in the reagent layer. In use, the spreading layer of elements according to U.S. Pat. No. 3,992,158 is contacted with an aqueous sample which distributes uniformly throughout the spreading layer and into the reagent layer. In the reagent layer, a material interacts with an analyte in the sample to produce a detectable change. This patent further discloses that the element may be readily adapted for use in the analysis of analytes such as albumin by appropriate choice of test reagents or other interactive materials. Neither BCG or similar reagents, nor the problem of interference to an analyte assay using chromogenic indicator reagent is disclosed in this patent.
Multizone elements for the determination of total protein are disclosed in U.S. Pat. No. 4,132,528 issued Jan. 2, 1979 to J. N. Eikenberry et al. Such elements are composed of a spreading zone for distributing analyte containing fluid and a reagent zone composed of reagent, an alkaline-providing composition, and an alkaline-protective polymer. Alkaline-protective polymers include poly(vinylpyrrolidone), poly(acrylamide), agarose and copolymers of vinylpyrrolidone and acrylamide. The reagent employed to quantitate total protein is a modified biuret composition which interacts indiscriminately with albumin and other proteins. Unlike the present invention which, in one embodiment, selectively determines albumin in the presence of protein interferents, the invention disclosed in U.S. Pat. No. 4,132,528 can only determine total protein by virtue of the reagent employed.